Individual subunits of the eukaryotic cytosolic chaperonin mediate interactions with binding sites located on subdomains of beta-actin

The chaperonin containing TCP-1 (CCT) of eukaryotic cytosol is composed of eight different subunit species that are proposed to have independent funct ions in folding its in vivo substrates, the actins and tubulins, CCT has be en loaded with S-35-beta-actin by in vitro translation in reticulocyte lysa te and then subjected to immunoprecipitation with all eight anti-CCT subuni t antibodies in mixed micelle buffers, conditions that disrupt CCT into its constituent monomers. Interactions between S-35-beta-actin and isolated CC T alpha, CCT beta, CCT epsilon, or CCT theta subunits are observed, suggest ing that polar and electrostatic interactions may mediate actin binding to these four CCT subunits, Additionally, a beta-actin peptide array was scree ned for CCT-binding sequences. Three regions rich in charged and polar amin o acid residues, which map to the surface of native beta-actin, are implica ted in interactions between actin and CCT. Several of these biochemical res ults are consistent with the recent cryo-electron microscopy three-dimensio nal structure of apo-CCT-alpha-actin, in which alpha-actin is bound by the apical domains of specific CCT subunits, A model is proposed in which actin interacts with several CCT subunits during its CCT-mediated folding cycle.